The overall goal of this application is to understand the role of the low molecular weight (LMW) thiol, bacillithiol (BSH), in bacterial physiology using a genetic and biochemical approach. LMW thiols play fundamental roles in numerous aspects of cell physiology, including response to reactive oxygen and electrophile stress, detoxification of antibiotics, host-pathogen interactions, and maintenance of metal ion homeostasis. The LMW thiol in the well-studied bacterium, Bacillus subtilis, has only recently been identified as BSH and its functions are poorly understood. In Aim 1, the role of BSH in the response to the reactive electrophiles, methylglyoxal (MG) and formaldehyde (FA) will be examined. MG and FA are endogenously produced products of metabolism. The accumulation of these toxic electrophiles can lead to DNA damage and protein inactivation and must be detoxified. Aim 2 will center on the contribution of BSH in cellular zinc homeostasis. Zinc ions are critical for essential cellular functions, serving structural roles in proteins and as cofactors for essential enzymes. Since zinc limitation and excess are both toxic to the cell, bacteria have evolved mechanisms to maintain the cytosolic zinc concentration within a narrow window. A fraction of the total zinc pool, termed the labile zinc pool, can be mobilized and redistributed in response to various signals to influence cellular function. BSH is proposed to play a major role in the maintenance of the labile zinc pool. The proposed work will provide a valuable perspective into the physiological roles and specialized functions of LMW thiols, such as BSH. It will also provide the first detailed description of the labile metal pool for any bacterium. Since Bacillus subtilis serves as the model organism for a number of medically and biotechnologically important Gram-positive bacteria, such as Bacillus anthracis, Staphylococcus aureus, and Listeria monocytogenes, the information generated from these studies will be of general interest.